The long term goal of this program is to delineate the pathway by which the vacuolar hydrolases of the primitive eukaryote Saccharomyces cerevisiae are processed and localized to the vacuole. Our specific aims are to isolate and characterize mutations which affect several vacuolar hydrolase activities simultaneously and to gain an understanding of the way in which the pleiotropic effect is achieved. By examination of the properties of doubly mutant strains in which one of the two mutations has been characterized as to the level or location within the pathway of the blockage, the order of mutations within the pathway can be determined. A second aim is to clone an study the genes and gene prodducts inactivated by the mutations, and to develop immunochemical reagents to allow monitoring of the levels and intracellular location of these gene products. A longer term goal is to understand what the function of these gene products is in the pathway.